Recent advances in the ability to construct arrays of biological molecules has greatly facilitated the ease and speed with which certain biological assays can be performed. For example, in the areas of nucleic acid sequencing and analysis, the advent of new technologies for constructing arrays of immobilized target nucleic acids or oligonucleotide probes has enabled the rapid screening and sequencing of nucleic acids. Arrays of peptides and small biomolecules have also proven useful in binding assays used in pharmaceutical development. The usefulness of these arrays depends on the ability to generate arrays with spatially addressable regions of defined composition or sequence.
Several technologies have been developed for producing these arrays of biological molecules. Several researchers have devised methods for in situ synthesis of arrays of biological polymers, such as nucleic acids, peptides, and carbohydrates. These methods use, for example, physical barriers to separate regions, devices (such as inkjet printers) for precise delivery of reagents to regions, or masking techniques that allow the use of light to determine the course of synthesis. See, e.g., WO 90/03382; Fodor et al., 1991, Science 251:767-73; Pease et aL., 1994, Proc. Natl. Acad. Sci. 91:5022-26; U.S. Pat. No. 5,424,186, to Fodor et al. Alternatively, presynthesized biomolecules or biological polymers may be attached directly to the substrate at precise positions using a variety of techniques, ranging from simple spotting to robotic delivery systems. A variety of different substrates and techniques for attaching the biomolecules to the substrates are also available.
As noted above, arrays of nucleic acids have proven particularly valuable. The ability to perform many previously available techniques has been greatly enhanced by availability of arrays, which permit many assays to be performed simultaneously on a single array rather than having to do each assay individually. Other techniques that would have been virtually impossible are now possible using polynucleotide arrays.
One technique that has been particularly enhanced by the availability of arrays of nucleic acids is sequencing by hybridization (SBH). SBH is a technique for rapidly sequencing nucleic acids without using gels. In SBH, polynucleotides having overlapping sequences are hybridized to a target nucleic acid. The sequences of the polynucleotides that hybridize are then determined and the common sequences overlapped to generate the sequence of the nucleic acid. The use of arrays has allowed the generation of sufficient hybridization information to make SBH feasible on a large scale.
SBH is divided into three formats, depending on the nature of the array and the way in which it is interrogated. In Format I, the target nucleic acid is immobilized and the labeled polynucleotides are in solution. In Format II, the polynucleotides are immobilized and the labeled target nucleic acid is in solution. In Format III, immobilized polynucleotides are hybridized with an unlabeled target nucleic acid and labeled oligonucleotide probes. Hybridization is assayed by ligating the labeled oligonucleotide probes to the immobilized polynucleotides. All three formats require the ability to distinguish perfectly matched hybrids from hybrids that contain a single mismatch at any position. For a more detailed discussion of SBH and the three formats, see WO 98/31836, particularly at pages 1-3.
While the demand for biological arrays, and in particular polynucleotide arrays, is high, current methodologies for constructing such arrays still suffer from certain difficulties. The most common difficulty is assaying the quality and integrity of an array once it has been fabricated. While the chemistries involved in producing the arrays are relatively well understood, methods for synthesizing arrays still suffer from lack of reliability and reproducibility, and even failure. However, identifying regions of attachment failures is very difficult, particularly with the small spots found in miniaturized arrays. Thus, quality control of produced arrays is very difficult to maintain. Furthermore, even minor variations in attachment efficiencies can make interpretation of results generated from such arrays very difficult, as the researcher may not be able to tell whether a difference in signal is real or merely an artifact of the attachment process. This problem is particularly acute in applications such as sequencing by hybridization, which require extremely accurate differentiation of even minor differences in hybridization.